Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder which is caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). It is characterized by progressive movement disorder, cognitive decline and psychiatric disturbances. Death ensues approximately 20 years after onset of the disease. No effective therapy currently exists for HD. HTT is a soluble protein of ~3,144 amino acids that has no sequence homology with other proteins. Increasing evidence indicates that HTT functions as a molecular scaffold that is able to organize a variety of signaling complexes. It is expressed ubiquitously in humans and rodents, with the highest levels found in CNS neurons and the testes. Because of the involvement of a single causal gene and simple genetic testing, HD and other diseases caused by CAG-repeat expansions offer unique therapeutic opportunities. The HD mutation is considered to be a "gain of function" mutation. Therefore, therapeutic approaches that are based on reducing mutant HTT expression such as RNA interference are currently considered to be promising strategies for HD treatment. It is likely that these agents will cause inactivation or impair normal function of both mutant and wild type HTT alleles. Therefore, monoallelic therapies targeting only the pathological allele are of particular interest. We propose to conduct proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is an efficient means to repair specifically the expanded, pathological allele, without functionally affecting the normal allele. This technology will restore not only the expression of the corrected mRNA from the dominant gene but also the production of the corresponding protein. Advantages of this technology include the possibility of utilizing small corrective RNA sequences that target exonic sequences within the mutated gene with high specificity as well as the natural regulation of gene expression. In specific aim 1 we will generate and functionally optimize trans-splicing constructs. The efficiency of these constructs will be tested using a binary cell transfection system, consisting of the trans-splicing module and an HTT minigene. In specific aim 2, selected optimized trans-splicing constructs will be further tested for the ability to repair endogenous HTT pre-mRNA. In addition, we will assess restoration of cellular functions as a result of successful HTT repair. Future work will be aimed at transferring the most efficient constructs into HD animal models. The knowledge gained in this project might eventually lead to a novel gene therapy for HD. PUBLIC HEALTH RELEVANCE: Huntington's Disease is a progressive and deadly neurodegenerative disease for which there is no cure. An inherited mutation causes the death of neurons in the brain. This project explores the possibility of repairing the mutation and to restore normal cellular functions.